Yeast artificial chromosomes (YACs) are used as a framework for physical mapping, but are poor substrates for gene identification techniques such as exon-amplification. To take advantage of YACs without necessarily having to use smaller vectors, we have developed a two-part strategy combining yeast artificial chromosome (YAC)-mediated gene transfer and representational difference analysis (RDA) whereby transcripts expressed from the transferred YAC are readily identified. The usefulness of this approach was assessed using resources developed during the positional cloning of NPC1, the gene responsible for Niemann Pick Type C (NP-C) (Science 277: 228-231, 1997). In the process of identifying NPC1, the candidate interval was substantially narrowed by complementation of the NP-C phenotype by stable integration of a 590kb YAC (911D5) into the genome of CT60, a hamster NP-C cell line (PNAS 94:7378, 1997). Given that NPC1 expression was detected only in complemented CT60 cell lines, we hypothesized that by using RDA, human genes (including NPC1) expressed from YAC 911D5, could be differentiated from endogenous hamster transcripts. We performed RDA using complemented CT60 clones as testers, and a non-complemented CT60 clone containing an integrated CFTR YAC as the driver. The difference products represented fragments of transcripts differentially expressed after YAC transfer, including NPC1, as indicated by Southern hybridization using the NPC1 cDNA. Non-NPC1 difference products were sequenced and analyzed by data base comparison and PCR mapping back to YAC 911D5. Two novel cDNA fragments expressed from 911D5 were identified along with cDNA fragments from genes not present on 911D5 which mapped to the CT60 genome. Thus, using NP-C as a model system, we have demonstrated that RDA is a useful method for isolating and identifying genes on YACs consisting of several hundred kilobases of DNA. The identification of novel non-NPC cDNA fragments suggests that RDA combined with YAC transfer to mammalian cells could be useful for gene identification when only a YAC physical contig exists, without monitoring phenotypic changes. The current strategy is advantageous because it accelerates the searching process. We are comparing the efficiency of this technique with common techniques and we are also attempting to adapt this strategy to identify other disease genes such as the gene responsible for inherited melanoma.